1. Field of the Invention
The present invention relates to extreme thermophile single-stranded DNA binding mutant proteins, and methods of use thereof. More specifically, the invention relates to extreme thermophile single-stranded DNA binding mutant proteins that can improve the amplification efficiency of a template nucleic acid in an isothermal amplification system that uses a strand displacement polymerase and methods of use thereof.
2. Description of the Related Art
Various methods for exponentially amplifying nucleic acids have been developed previously, and the methods that amplify nucleic acids particularly efficiently can generally be separated into those that use a thermal cycle in which the reaction temperature fluctuates and those in which the reaction temperature remains constant.
Polymerase chain reaction, that is, PCR (see Non-Patent Document 1, for example), is known as one method that uses a thermal cycle. In PCR, two primers that have a base sequence complementary to a target nucleic acid template are mixed with the template nucleic acid. Next, normally a single cycle involving denaturation of the template nucleic acid, annealing the primers to the template nucleic acid, and extension of the primers by DNA polymerase (DNA replication) is performed for twenty to thirty cycles in order to synthesize a strand complementary to the template nucleic acid between the two primers that have been annealed to the template nucleic acid. In this method, the synthesized strand can serve as a new template nucleic acid, and thus the template nucleic acid can be amplified exponentially through replication in other cycles using the same primer set. To withstand the elevated temperatures that are required to denature the template nucleic acid in each cycle, it is necessary to use a heat-stable DNA polymerase. Further, in DNA amplification by PCR, the amplification reaction does not proceed in a continuous manner and thus the nucleic acid sample, that is, the template nucleic acid, must be supplied over a series of a plurality of cycles as amplification is carried out.
By contrast, strand displacement amplification (SDA) (for example, see Non-Patent Document 2) and rolling circle amplification (RCA) (for example, see Non-Patent Documents 3, 4, and 5) are known as methods in which the template nucleic acid amplification reactions are performed isothermally. In SDA, a restriction enzyme nicks a template nucleic acid and the action of a DNA polymerase (strand displacement polymerase), which one by one displaces these DNA fragments having nicks, is used to amplify the DNA. RCA, on the other hand, involves displacement by a strand displacement polymerase of the strand before the tip of an elongation strand that has been synthesized, with using, as its origin, a primer annealed to a template nucleic acid in order to produce a hybrid. These methods therefore do not require a thermal cycle because amplification of target DNA sequences is carried out isothermally in a continuous manner.
Such strand displacement allows template nucleic acids to be linearly or exponentially amplified in a continuous manner under isothermal conditions. Consequently, some advantages to RCA, for example, include that it can more efficiently increase the amount of amplification product that is produced because the procedure of template nucleic acid amplification is simpler than methods that use a thermal cycle, that there are no limitations regarding the length of template nucleic acids that can be amplified effectively, and that equipment for performing the thermal cycle is not necessary.
Here, in the template nucleic acid amplification reaction, single-stranded DNA binding protein (hereinafter may also be abbreviated as “SSB protein”) is known to be related to the efficiency of the template nucleic acid amplification reaction, for example.
SSB protein has high affinity for single-stranded DNA (ssDNA) in a manner that is not sequence specific. Normally, SSB protein is necessary or DNA replication and recombination and for repair of the organism genome. SSB protein specifically stimulates homologous DNA polymerase, increases the fidelity of DNA synthesis, improves the ability of DNA polymerase to advance forward by destabilizing the helix and promotes DNA polymerase binding, and organizes and stabilizes the replication origin. That is, SSB protein is known to act as a replication assisting protein (for example, see Patent Documents 1 and 2).
Numerous examples of SSB proteins have been isolated from a wide array of sources ranging from bacteriophages to eukaryotes. For example, Patent Document 1 discloses the replication protein A-1 (rpa-1) from beer yeast (Saccharomyces cerevisiae), a mitochondrial replication protein (rim-1), the bacteriophage T7 gene 2.5 protein (gp 2.5), the bacteriophage phi29 protein p5 (p5), the T4 gene 32 protein (gp32), and the Escherichia coli SSB protein. It is also known that SSB protein has been isolated from extreme thermophiles as well (for example, see Non-Patent Documents 6 and 7).
Patent Document 1 describes the addition of SSB protein to an isothermal amplification reaction system in order to improve the efficiency of template nucleic acid amplification. In Patent Document 2, E. coli SSB protein is used as an effective strand displacement factor for the strand displacement replication of a template nucleic acid. That is, it discloses that, in the presence of the strand displacement factor, a strand displacement polymerase that can carry out strand displacement replication (such as the bacteriophage phi29 DNA polymerase) is used to amplify template nucleic acid through RCA.
These template nucleic acid amplification methods that use strand displacement polymerases are dependent on the strand displacement ability of that strand displacement polymerase, which denatures the template nucleic acid. Since strand displacement can be promoted by replication assisting proteins and strand displacement factors, it was thought that the presence of replication assisting proteins or strand displacement factors would allow DNA fragments that are specific to the template nucleic acid to be amplified efficiently.    Patent Document 1: JP H10-284889A (see paragraphs 0007 and 0014, for example)    Patent Document 2: JP 2002-525078A (see paragraphs 0059 to 0062, for example)    Non-Patent Document 1: Saiki et al., Science 230:1350-1354, 1985    Non-Patent Document 2: Walker et al., Proc. Natl. Acad. Sci. USA 89: 392-896, 1992    Non-Patent Document 3: Amersham Biosciences, “Product Catalog: GenomiPhi DNA Amplification Kit,” online, searched on the Internet on Apr. 28, 2005, <URL: http://www.jp.amershambiosciences.com/catalog/web_catalog.asp?frame5_Value=912&goods_name=GenomiPhi+DNA+Amplification+Kit>    Non-Patent Document 4: Dean et al., Genome Res. 11(6), 1095-1099, June 2001    Non-Patent Document 5: Lizardi et al., Nature Genetics 19(3), 225-282, July 1998    Non-Patent Document 6: Dabroski et al., Microbiology, 148(Pt10), 3307-3315, October 2002    Non-Patent Document 7: Perales et al., Nucleic Acids Research, 31(22), 6473-6480, November 2003
However, in isothermal amplification systems that use a strand displacement polymerase there is the problem that, although DNA fragments that are specific to the template nucleic acid amplified efficiently, DNA fragments that are non-specific to the template nucleic acid also are easily amplified as well. The methods disclosed in Patent Documents 1 and 2 involve adding SSB protein from E. coli or yeast to carry out the isothermal amplification reaction, and in these methods as well there was the problem that it was not possible to inhibit the amplification of non-specific DNA fragments.
A possible reason or this is that the temperature during isothermal amplification normally is about 30 to 60° C. and thus primer dimers readily form, and these primer dimers that have formed increase the likelihood that DNA fragments that are non-specific for the template nucleic acid will be amplified. In other words, primer dimers are formed even when a template nucleic acid is not present and lead to the amplification of non-specific nucleic acids. DNA fragments that are non-specific for a template nucleic acid lower the amplification precision and become background noise that impedes later experiments. Since primers with random sequences are used in the amplification, it was believed to be difficult to control non-specific amplification.
For these reasons, isothermal amplification methods for template nucleic acids have the potential to become widespread due to the fact that they do not require a thermal cycle like PCR, for example, but at the present time they have found only limited application due to problems with the amplification precision resulting from the non-specific amplification discussed above.